Erythromycin is a potent antibiotic with current annual production of some 2,200 tons. This clinically useful, broad-spectrum macrolide antibiotic is naturally produced by the actinomycetes Saccharopolyspora erythraea. In spite of extensive classical strain development efforts, classical mutagenesis and selection, volumetric yields of erythromycin in fermentation processes remain rather low at 8 to 9 g/l, as compared to 5-10 fold higher yields in the case of penicillins. Hence, production costs remain high.
Genetic engineering is one approach to overcome the limitations of classical strain improvement programs (see, for review, Lal et al., 1996, Crit. Rev. Microbiol. 22(4):201-255). Thus far, recombinant DNA techniques with erythromycin-producing strains of S. erythraea have been attempted on only a few strains. The approach generally used has been to identify and clone the genes encoding antibiotic biosynthetic proteins, so as to modify or amplify them.
For example, Hanel et al. attempted to achieve higher levels of erythromycin A production in S. erythraea by inserting into the chromosome an extra copy of eryC1, a gene presumably involved in directly regulating expression of erythromycin biosynthetic genes. Hanel et al., 1993, Biotech. Lett. 15:105-110. In the presence of thiostrepton, the selectable marker gene used for transformation, erythromycin A production of the transformants was two to three fold higher than the non-transformed strain. However, this increased production was lost in the absence of thiostrepton.
Other approaches that have been advanced to increase production levels of erythromycin have been to clone and amplify the biosynthetic erythromycin gene cluster (Lal et al. supra.), or to transfer the erythromycin biosynthetic gene cluster into another organism for potentially higher production. Kao et al., 1994, Science 265:509-512.
Production of some antibiotics is highly dependent on the amount of oxygen available during culture conditions. Clark et al., 1995, Microbiology 141:663-669. Accordingly, one way of increasing production of these types of antibiotics has been to engineer the microbial host to express the Vitreoscilla hemoglobin gene (VHb). Such a metabolic engineering strategy has been shown effective in increasing actinorhodin and cephalosporin C production in Streptomyces coelicolor and Acremonium chrysogenum, respectively (Magnolo, S. K. et al., 1991, Bio/Technology 9:473-476; DeModena, J. A. et al., 1993, Bio/Technology 11:926-929). At low dissolved oxygen levels (DO below 5% of air saturation), S. coelicolor transformed with the VHb gene produced ten fold more actinorhodin than non-transformed S. coelicolor. Magnolo et al., supra. However, when oxygen was not limiting (DO greater than 40% air saturation), both transformed and non-transformed strains produced similar amounts of antibiotic. Id. Production of cephalosporin C by the filamentous fungi A. chrysogenum is also severely reduced under low oxygen conditions. DeModena, J. A. et al., supra. Cultures of transformants expressing high levels of VHb yielded higher amounts of cephalosporin C, especially under oxygen limited conditions.
In contrast to these and many other antibiotics, erythromycin production does not appear sensitive to the levels of dissolved oxygen during culture. Heydarian et al., 1996, Biotechnol. Letts. 18:1181-1186; Clark et al., 1995, Microbiol. 141:663-669. Heydarian et al. reported that although growth of S. erythraea cultures is inhibited at a low constant dissolved oxygen tension (DOT) of 10% air saturation, the specific erythromycin production is virtually identical to that of a culture where the DOT did not fall below 65%. Similarly, Clark et al. found that erythromycin was produced in both oxygen limited and oxygen sufficient cultures. At the same time, however, Clark et al. discovered that a different actinomycete, Amycolatopsis orientalis, produced the antibiotic vancomycin only in oxygen-sufficient cultures. The results for both species were comparable in both shake flasks and bioreactors. Accordingly, availability of oxygen was not considered a critical limitation to erythromycin production, as long as the dissolved oxygen concentration in culture are above minimal levels required for growth.